This proposal concerns the regulatory GTPases Ran and Rho, which are involved in the control of polarized cell behavior and nuclear import. Novel fluorescent protein analogs will be designed to report the nucleotide state of these small GTPase signalling proteins within individual, living cells. Alternate approaches will be developed to enable production of such analogs based on a wide variety of GTPases, and these methods will be applied here to produce indicators of Ran and Rho family GTPases. Because small GTPases are ubiquitous and impact diverse fundamental cell behaviors, the new technology validated in these studies will be of broad utility and significance. A full understanding of Rho family and Ran GTPase function requires knowledge of the spatial organization of signalling within intact cells. The nucleotide state indicators will be used to study the spatial and temporal dynamics of GTPase activation in living cells. The mechanisms by which Rho GTPases control cell polarization and motility will be examined in wound healing fibroblasts by characterizing changes in subcellular localization, the spatial and temporal dynamics of activation, and by correlating these behaviors with the dynamics of the cytoskeleton. Both the location and levels of activation will be quantified. Hypotheses regarding modulation of integrin and growth factor crosstalk through GTPase localization and variation of activation levels will be explored. In studies of Ran function, the indicators will reveal the nucleotide state of Ran in different subcellular locations, distinguishing between potential mechanisms for regulation of nuclear import rate and cell cycle feedback controls linking progression of mitosis to DNA replication. The fundamental processes controlled by these GTPases, including cell motility, nuclear import, and regulation of cell morphology, are critical to normal homeostasis and relevant to many diseases and immune function.